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Monocyte-Derived Plasminogen Activator Inhibitor (Serine Protease Inhibitor/Urokinase/Cdna Cloning/Bacterial Expression) T

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Monocyte-Derived Plasminogen Activator Inhibitor (Serine Protease Inhibitor/Urokinase/Cdna Cloning/Bacterial Expression) T Proc. Natl. Acad. Sci. USA Vol. 85, pp. 985-989, February 1988 Biochemistry Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor (serine protease inhibitor/urokinase/cDNA cloning/bacterial expression) T. M. ANTALIS*t, M. A. CLARK*, T. BARNES*, P. R. LEHRBACH*, P. L. DEVINE*, G. SCHEVZOV*, N. H. Goss*, R. W. STEPHENSt§, AND P. TOLSTOSHEV* *Biotechnology Australia Pty. Ltd., P. 0. Box 20, Roseville, New South Wales 2069, Australia: and tDepartment of Medicine and Clinical Science, Australian National University, Woden Valley Hospital, Garran, Australian Capital Territory 2605, Australia Communicated by Harland G. Wood, October 8, 1987 ABSTRACT Human monocyte-derived plasminogen acti- similar specificity to the placental PAI (7), particularly in the vator inhibitor (mPAI-2) was purified to homogeneity from the presence of fibrin (12). U937 cell line and partially sequenced. Oligonucleotide probes The monocyte-derived PAI (mPAI-2) was originally de- derived from this sequence were used to screen a cDNA library scribed by Golder and Stephens (6) and termed minactivin. prepared from U937 cells. One positive clone was sequenced We have defined this molecule as a PAI-2 type¶ on the basis and contained most of the coding sequence as well as a long of its immunological crossreactivity with the placental PAI. incomplete 3' untranslated region (1112 base pairs). This We designate this molecule mPAI-2. A convenient source of cDNA sequence was shown to encode mPAI-2 by hybrid-select the mPAI-2 is the human histiocytic lymphoma cell line translation. A cDNA clone encoding the remainder of the U937 (7). mPAI-2 mRNA was obtained by primer extension of U937 We report the cloning and sequencing of the cDNA coding poly(A)+ RNA using a probe complementary to the mPAI-2 for mPAI-2 from the U937 cell line11 and its expression in coding region. The coding sequence for mPAI-2 was placed Escherichia coli cells. The deduced amino acid sequence under the control of the A PL promoter, and the protein reveals marked homology to the serine protease inhibitor expressed in Escherichia coli formed a complex with urokinase (serpin) superfamily (13) and, in particular, appears to have that could be detected immunologically. By nucleotide se- several striking features in common with the chicken oval- quence analysis, mPAI-2 cDNA encodes a protein containing bumin gene family. 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows MATERIALS AND METHODS extensive homology with members of the serine protease Assays. mPAI-2 activity was measured by a modification inhibitor (serpin) superfamily. mPAI-2 was found to be more (14) of the method of Coleman and Green. Human urokinase homologous to ovalbumin (37%) than the endothelial plasmin- was purchased from Calbiochem-Behring. Plasminogen was ogen activator inhibitor, PAI-1 (26%). Like ovalbumin, purified from fresh human plasma by lysine-Sepharose mPAI-2 appears to have no typical amino-terminal signal (Pharmacia) affinity chromatography (15). Endoproteinase sequence. The 3' untranslated region of the mPAI-2 cDNA Lys-C was purchased from Boehringer Mannheim. Affinity- contains a putative regulatory sequence that has been associ- purified antibodies to placental inhibitor (16) that crossre- ated with the inflammatory mediators. acted with mPAI-2 were supplied by B. Astedt (University of Lund, Sweden). Plasminogen activators (PA) are serine proteases that medi- Purification of mPAI-2 and Partial Amino Acid Sequence. ate proteolytic cascades involved in cellular translocation, mPAI-2 was purified from the conditioned medium (2 liters) migration, and invasion. These are generally of two types: of U937 cells (American Type Culture Collection CRL 1593) tissue type (tPA), with a high affinity for fibrin and likely the grown in the presence of 4-phorbol 12-myristate 13-acetate major PA in the fibrinolytic system, and urokinase type (PMA) at 30 ng/ml. Concentrated culture supernatants were (uPA), associated with extracellular proteolytic events such applied to a phenyl-Sepharose column equilibrated with 50 as tissue remodeling and destruction, particularly in the mM sodium citrate/2 M NaCI, pH 5.5, and washed with 50 invasive growth and metastatic spread of malignant tumors mM sodium citrate, pH 5.5/0.5 M NaCl/1 mM EDTA, and (reviewed in ref. 1). the mPAI-2 was eluted in 50 mM glycine at pH 9.0. After Regulation of the activity of PA involves specific inhibi- fractionation on Sephacryl S-200, the mPAI-2 preparation tors. These have been classified into four immunologically was electrofocused with a pH gradient of 4.5-6.0. Pure distinct groups: PAT-1 type PA inhibitor from endothelial mPAI-2 was then obtained by reverse-phase (Vydac C4; cells (2, 3); PAI-2 type PA inhibitor from placenta (4, 5), Separations Group, Hesperia, CA) high-pressure liquid monocytes (6), and macrophages (7); the recently described chromatography using a gradient of acetonitrile in 0.1% urinary inhibitor (8); and protease nexin-I (9). The first three PAI are fast acting and specific for PA, whereas protease Abbreviations: PA, plasminogen activator(s); PAI, PA inhibitor(s); nexin-I preferentially inhibits thrombin and has a secondary mPAI-2, monocyte-derived PAI-2; tPA, tissue-type PA; uPA, uro- kinase-type PA; PMA, 4-phorbol 12-myristate 13-acetate. action on plasminogen activation. Endothelial PAI effi- tTo whom reprint requests should be addressed. ciently inhibits tPA and uPA (10), whereas the placental PAI §Present address: Department of Virology, University of Helsinki, is a potent inhibitor of uPA and reacts to a much lesser Haartmaninkatu 3, 00290 Helsinki 29, Finland. extent with tPA (11). The monocyte-derived PAI has a ¶The nomenclature used in this paper is that recommended by the 32nd International Committee on Thrombosis and Hemostasis, June 8, 1986, Jerusalem, Israel. The publication costs of this article were defrayed in part by page charge 1This sequence is being deposited in the EMBL/GenBank data base payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03603). 985 986 Biochemistry: Antalis et al. Proc. Natl. Acad. Sci. USA 85 (1988) trifluoroacetic acid (CF3COOH). Following digestion of tants derived from PMA-stimulated U937 cells. The protein mPAI-2 (4 gg) with endoproteinase Lys-C (0.1 Ag) in 20 mM migrated as a single band having a pI of 5-5.2 and Mr of Tris HCI, pH 8.5/5 M urea for 8 hr at 22TC, the resultant 45,000-48,000. It was not possible to derive amino acid peptides were separated by reverse-phase (Snychropak RP- sequence from the intact molecule as the amino terminus of P-C8; SynChrom, Linden, IN) high-pressure liquid chroma- the purified protein was blocked, as has been observed by tography using a gradient of acetonitrile in 0.1% CF3COOH. others (7). Partial amino acid sequences were therefore Partial amino acid sequence was determined by using an determined from selected peptides obtained from an endo- Applied Biosystems (Foster City, CA) 470A gas-phase se- proteinase Lys-C digest of mPAI-2 as described in Materials quencer. The complete details of the purification of mPAI-2 and Methods. These are as follows: peptide will be published elsewhere. 13, Ala-Gln-Ile- Construction of cDNA Libraries. A AgtlO cDNA library was Leu-Glu-Leu-Pro-Tyr-Xaa-Gly-Asp-Val-Xaa-Met-Phe-Leu- constructed with RNA extracted by a modified guanidine HCl Leu-Leu-Pro-Xaa-Glu; peptide 11, Gly-Arg-Ala-Asn-Phe- method (17) from U937 cells that had been cultured in the Ser-Gly-Met-Ser-Glu-Xaa-Asn-Asp-Leu-Phe; peptide 10, presence of PMA at 30 ng/ml for 19 hr. Poly(A) + mRNA was Met-Ala-Glu-Xaa-Glu-Val-Glu-Val-Tyr-Ile-Pro-Gln-Phe- isolated by using oligo(dT)-cellulose (18). Double-stranded Lys-Leu-Glu-Glu-Xaa-Tyr; peptide 6, Leu-Asn-Ile-Gly-Tyr- cDNA was synthesized essentially by the S1 nuclease method Ile-Glu-Asp-Leu-Lys; peptide 9, Ile-Pro-Asn-Leu-Leu-Pro- (19). Following methylation and ligation of EcoRI linkers, Glu-Gly-Xaa-Val. cDNA was fractionated and cDNAs >1000 base pairs (bp) Identification of mPAI-2 Translation Product. In vitro were ligated to phage AgtlO arms, packaged by using a translation of U937 mRNA followed by immunoprecipitation Gigapack (Vector Cloning Systems, San Diego, CA) packag- with anti-placental inhibitor antibodies yielded a translation ing extract, and plated on E. coli C600 lft. The cDNA library product of Mr 43,000-45,000 that was biologically active, as contained -1.5 x 106 independent recombinants. A second evidenced by a shift in mobility in the presence of urokinase library was constructed by 3' extension of U937 mRNA by (Mr 33,000) to Mr 69,000 characteristic of the formation of a using a synthetic oligonucleotide primer derived from the 5' urokinase-mPAI-2 complex (Fig. 1). region of the partially sequenced gene: 5' TTC CAG TAA Isolation of PAI-2 cDNA. The primary cDNA library was ATA ATT CCC TGT GGA TGC ATT 3' (complementary to screened with each of the oligonucleotide probes and cloned nucleotides 391-420; see Fig. 2). Double-stranded cDNA was sequences that hybridized to more than one of the probes synthesized by using the RNase H method (20) and the library were selected for further study. The clone containing the constructed in AgtlO as described above. largest cDNA insert was analyzed by hybrid-select transla- Screening of cDNA Libraries.
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